(a) Field of the Invention
The present invention relates to a method for production of corosolic acid comprising the steps of suspension culturing plants cells in medium for cell growth, and suspension cell culturing at a high concentration in medium for cell production with adding an inducing agent.
(b) Description of the Related Art
Corosolic acid represented by the following Structural Formula I is a material present in leaves of Banaba which belongs to Lythraceae, and leaves of Loquat and flesh of Hawthorn which belong to Rosaceae. It has been reported that corosolic acid has the activity of promoting a rapid absorption of glucose into cells, similar to that of insulin (Chem Pharm. Bull. 41(12): 2129-2131, 1993). Further, it has also been shown from clinical demonstration that corosolic acid can rawer blood-sugar levels without any side effects and prevent the re-rise of blood-sugar levels (Journal of Ethnopharmacology 87: 115 117, 2003). Due to such activity, corosolic acid has been employed as a sugar control agent and a therapeutic agent against Type 2 diabetes (U.S. Pat. No. 6,485,760), as well as a weight control agent (U.S. Pat. No. 6,784,206).

At present, corosolic acid is obtained by hot water extraction and alcohol extraction techniques from leaves of Banaba. However, since the amount of corosolic acid naturally present in the leaves is very small, supply of corosolic acid through the direct extraction from natural plants is significantly limited, and thereby, does not meet the increased demand for corosolic acid as raw materials of health foods and medicines. On the other hand, the methods to produce corosolic acid using a semi-synthetic technique from ursolic acid which is a precursor of corosolic acid, has been reported. However, this method also has economic disadvantages because the processes of natural extraction and chemical synthesis should be involved.
Plants serve as sources of many useful secondary metabolites such as alkaloids, steroids, terpenoids and phenol-based compounds which are used in medical materials, dyes, pigments, spices, food additives, etc. Such useful metabolites are currently produced by direct extraction of cultivated plants and purification. However, such conventional processes have the problem of to being greatly affected by geographical and climatic conditions, and policies, as well as change of cultivation environments. Since the content of useful metabolites in plants is not sufficiently high, and most of the plant-originated useful materials produced have complicated structures having been synthesized through various stages of biosynthesis pathways, it is disadvantageous to produce the materials through direct extraction or chemical synthesis methods. Therefore, there has been continuous research to develop methods of mass production of secondary metabolites through plant cell culture techniques.
In spite of a lot of advantages inherent in plant cell culture techniques, low productivity and instability of productivity are considered to be the major hurdles that prevent the techniques from being readily applied to relevant industry. Although it is relatively easy to induce dedifferentiated callus tissues from plant tissues, such induced callus tissues themselves are difficult to apply in relevant industry. Therefore, it is necessary to induce a suspension-culture cell line which can be cultured in liquid culture medium. However, in this regard, it is very difficult to select and establish a superior cell line which can produce secondary metabolites in large amounts and maintain such a high productivity. Further, the steps of selecting and establishing the suspension-cultured cell lines require significant time and effort. Nevertheless, further procedures such as optimizing the medium composition and culturing conditions, selecting and applying, proper elicitors, and developing culturing techniques capable of increasing the productivity, should be required for establishing a mass production system using the selected and established cell lines.
Results of inducing callus tissues from plant bodies of Loquat, and examining the level of production of corosolic acid therefrom have been reported (Taniguchi S. et al., Phytochemistry 59: 315 323, 2002), however the results show that the level of the produced corosolic acid is similar to that in the natural leaves at most. However, there has been neither report nor suggestion regarding the induction and establishment of suspension-cultured cell lines, which is essential for applying the plant cell culture technique to the relevant industry, and a method of mass production of corosolic acid. Likewise for Banaba, there have been no reports regarding the induction and establishment of suspension-cultured cell lines, and a method of mass production of corosolic acid.